Reporter assay is a means for quantifying the transcriptional activity of a transcriptional regulatory sequence. Reporter assay is carried out by ligating a gene (hereinafter, referred to as “reporter gene”) encoding a reporter protein under the control of a transcriptional regulatory sequence (e.g., promoter and enhancer) to be examined, introducing the resultant into a host cell, and then causing the expression of the protein. At this time, the transcriptional activity of a promoter is positively correlated with the amount of reporter protein generated by transcription and translation, for example. Hence, through quantification of the amount of reporter protein, the degree (high or low) of the relative transcriptional activity of a promoter can be evaluated.
Reporter assay can be carried out using various proteins as reporter proteins. For example, when a fluorescent protein is used as reporter protein, the thus expressed fluorescent protein is irradiated with excitation light and then the thus generated fluorescence intensity is measured, so that the relative amount of the reporter protein can be quantified (this method is referred to as a fluorescence method).
Furthermore, for example, reporter assay can be carried out using an enzyme such as β-galactosidase or alkaline phosphatase as a reporter protein. When an enzyme is used as a reporter protein, the relative amount of the reporter protein can be quantified by colorimetry with the use of a substrate that is degraded by the action of the enzyme so as to generate a color substance (this method is referred to as calorimetric method). Another method involves the use of a substrate that causes the generation of a luminescent substance instead of a substrate that causes the generation of a color substance. In this case, the relative amount of reporter protein can be quantified through measurement of the amount of luminescence (this method is referred to as a luminescent method).
Such a luminescent method has the following excellent characteristics. First, the method does not require any excitation light unlike a fluorescence method, so that the background is small and a high ratio of signal to noise can be obtained. Furthermore, the method has high sensitivity, by which a broad dynamic range can be obtained. Moreover, the method is excellent in terms of quantitative capability.
An example of an enzyme reaction system that is generally used in the luminescent method is a luciferase/luciferin reaction system.
Various types of luciferase are known, and they differ significantly from each other in terms of primary structure. For example, there are various luciferases derived from various organisms, including firefly and Renilla. 
Meanwhile, various types of luciferins are present as substrates, which differ greatly from each other in terms of chemical structure.
Types of luciferin that each luciferase recognizes as a substrate are limited to some extent. A technique generally referred to as a dual reporter assay involves adding a luciferin derived from firefly (hereinafter, referred to as “firefly luciferin”) and a luciferin derived from Renilla (hereinafter, referred to as “Renilla luciferin”) (coelenterazine) successively to a sample solution containing a mixture of a luciferase derived from firefly (hereinafter, referred to as “firefly luciferase”) and a luciferase derived from Renilla (hereinafter, referred to as “Renilla luciferase”) and then separately measuring the activity of the firefly luciferase and the activity of the Renilla luciferase.
Sea-firefly includes species such as Vargula hilgendorfii and Cypridina noctiluca. In such species of sea-firefly, luciferase is released ex vivo (specifically, in sea water) and then luciferin reacts with oxygen in sea water because of the catalytic action of the luciferase so as to produce luminescence.
Genes encoding a luciferase derived from Vargula hilgendorfii (hereinafter, referred to as “VLuc”) and a luciferase derived from Cypridina noctiluca (hereinafter, referred to as “CLuc”), respectively, have been cloned (Thompson, E. M., Nagata S., Tsuji F. I., “Proceedings of the National Academy of Sciences of the United States of America,” 1989, Vol. 86, p. 6567-6571; and Nakajima, Y., Kobayashi, K., Yamagishi, K., Enomoto, T., Ohmiya, Y., “Bioscience and Biotechnology and Biochemistry,” 2004, Vol. 68, p. 565-570). Both VLuc and CLuc are expressed in cultured cells and can be caused to be secreted extracellularly (JP Patent Publication (Kokai) No. 3-30678 A (1991) and International Publication No. 2006/132350 Pamphlet). Specifically, VLuc and CLuc are secretory luciferases. Therefore, such a gene encoding the luciferase (hereinafter, referred to as a “luciferase gene”) is used as a reporter gene, and the transcriptional activity of a transcriptional regulatory sequence such as a promoter can be measured without disrupting cells (International Patent Publication No. 2006/132350 Pamphlet).
In the case of secretory luciferases, a culture solution containing a secretory luciferase can be directly used as a solution to be tested. Hence, secretory luciferases are appropriate for construction of, namely, a high-throughput reporter assay system for treatment of many samples. On the other hand, in the case of non-secretory luciferases, collection of cells by centrifugation and disruption (or enhancement of cell permeabilization) of cells by ultrasonication, treatment with a surfactant, treatment with an organic solvent, or the like are essential. These procedures are inappropriate for treatment of numerous samples. Furthermore, in the case of secretory luciferases, a sample for measurement can be obtained by collecting a portion of a culture solution without disrupting cells. Thus, sampling can be carried out consecutively for the same cells. On the other hand, in the case of non-secretory luciferases, cells are always damaged by cell disruption or the like. Hence, consecutive sampling with the use of the same cells is impossible and as many different cells should be prepared as the number of measurement points.
It has been reported that the above CLuc is secreted in a culture solution at a level 320 times greater than VLuc, when expressed in NIH3T3 cells, and 410 times greater than VLuc, when expressed in HeLaS3 cells (Nakajima, Y., Kobayashi, K., Yamagishi, K., Enomoto, T., Ohmiya, Y., “Bioscience and Biotechnology and Biochemistry,” 2004, Vol. 68, p. 565-570). Therefore, compared with VLuc, CLuc is appropriate for use in a high-sensitivity, high-throughput reporter assay system using cultured cells as hosts.
Furthermore, a secretory high-throughput reporter assay system using a budding yeast Saccharomyces cerevisiae into which a CLuc gene has been introduced has also been conceived (International Publication No. 2006/132350 Pamphlet).
The luminescence mechanism of a luciferase/luciferin reaction system is generally considered to be as follows. First, luciferin is oxidized by catalytic action of luciferase into oxyluciferin in its excited state. Subsequently, oxyluciferin in its excited state immediately returns to the ground state, during which the oxyluciferin releases energy in the form of light (produces luminescence). The amount of luminescence produced per unit of time is thought to be proportional to the amount of luciferase existing in the system. Thus, the relative amount of luciferase can be quantified based on the luminescence.
With the above luminescence mechanism, luminescence can be obtained in accordance with the difference in energy level between the excited state and the ground state of oxyluciferin. A change in energy level difference appears as an emission spectrum change. Specifically, when the energy level in the excited state changes for some reason upon production of luminescence, luminescence with a color differing from that of a general case is obtained. This effect is known to take place due to a significant difference or a local difference in terms of the primary structure of luciferase (Viviani, V., Uchida, A., Suenaga, N., Ryufuku, M., Ohmiya, Y., “Biochemistry and Biophysics Research Communication,” 2001, Vol. 280, p. 1286-1291).
Meanwhile, there are at least two methods (referred to as “multi-reporter assay”) for simultaneously carrying out reporter assays of 2 or more types of promoter activity using a luciferase gene as a reporter gene, as follows.
A first method involves the use of a plurality of different chemical species of luciferins and luciferases having substrate specificity for each luciferin. In the case of this method, no reaction takes place with combinations other than the combination of a luciferase and a luciferin that form a pair, because of differences in substrate specificity. Furthermore, appropriate conditions (e.g., compositions of reaction solutions and hydrogen ion concentrations) differ depending on the reaction of each luciferase/luciferin reaction system. In the case of this method, reaction conditions should be varied depending on each luciferase/luciferin reaction, for one specimen, and the reactions should be carried out in order or in parallel. In accordance therewith, multiple luminescence measurements should be carried out for one specimen by employing different conditions appropriate for each luciferase/luciferin reaction. As described above, this method is problematic in terms of its complicated measurement procedures.
A second method involves the use of luciferins of the same chemical species as substrates. In this case, multiple types of luciferase whose substrates are luciferins of the same chemical species are used as reporter proteins. The amino acid sequences of these luciferases partially differ from each other, and they are characterized in that different emission spectra are generated from luciferases. The luminescence intensity originating from each luciferase should be determined and quantified based on differences in spectrum.
A multi-reporter assay using the above second method has the advantage of being simple because only a single type of substrate is used, and because the luminous reaction and the measurement can each be completed at one time.
In the case of luminescence simultaneously produced from luciferases with different luminescent colors, the spectra thereof may overlap. However, even under such circumstances, a method for estimating the luminescence intensity originating from each luciferase has been conceived (JP Patent Publication (Kokai) No. 2004-333457 A).
An example of a multi-reporter assay using the principle of the above second method is a method that involves the use of a luciferase gene derived from a luminescent beetle and a mutant gene thereof (Yoshihiro Nakajima and Yoshihiro Ohmiya, “Biotechnology Journal,” 2006, Vol. 6, No. 2, p. 230-232). However, such a luciferase derived from a luminescent beetle is non-secretory. Therefore, the luciferase is inappropriate for use in high-throughput reporter assays for the reasons as described above.
As described above, no high-throughput multi-reporter assay using the principle of the above second method is currently known. Furthermore, concerning sea-firefly luciferase, the presence of any mutant luciferase that alters luminescent color is unknown.
Meanwhile, a phenomenon referred to as BRET (Bioluminescence resonance energy transfer) is used as a method for detecting the structural changes of proteins at the biochemical level or the cellular level, for example (Otsuji, T., Okuda-Ashitaka, E., Kojima, S., Akiyama, H., Ito, S., Ohmiya, Y., “Analytical Biochemistry,” 2004, Vol. 329, p. 230-237).
In BRET, a luminous object and a fluorescing object form a pair. As a luminous object, a bioluminescent substance such as luciferase, luciferin, or the like is used. On the other hand, as a fluorescing object, for example, a chemical substance producing fluorescence or a fluorescent protein such as a green fluorescent protein (GFP) is used. When a luminous object and a fluorescing object are located at positions that enable (in terms of distance) topological energy transfer, the luminous object is excited and the energy released when it returns to the ground state is transferred to the fluorescing object. Subsequently the fluorescing object is excited and it emits light when it returns to the ground state. Each fluorescing object has its unique excitation spectrum and excitation efficiency is known to depend on the emission spectrum of the luminous object and the excitation spectrum of the fluorescing object. A luminous object emitting light at a wavelength that efficiently excites a fluorescing object is most preferable for composing an efficient BRET pair.
Hence, luciferase to be used in BRET analysis using luciferase and luciferin as luminous objects preferably emits light at a wavelength that efficiently excites the fluorescing object, with reference to the excitation spectrum of a fluorescing object to be used in the form of a pair. Therefore, the presence of mutant luciferases emitting light at different wavelengths makes it possible to form BRET pairs appropriate for various fluorescing objects.